1. Field of the Invention
The present invention relates to methods and apparatuses for sorting objects in forensic DNA analysis and medical diagnostics. More particularly, the present invention relates to the sorting of sperm for a single cell polymerase chain reaction (PCR) method which is used in forensic DNA analysis, such as STR analysis, to identify persons/assailants in sexual assault cases. Further, the present invention relates to the use of on-chip PCR in forensic DNA analysis. Finally, the present invention of sorting cells and single cell PCR has applicability outside of forensics, such as in the field of cancer diagnostics, where STR-based cell identification is becoming popular to distinguish cancer cells from healthy cells.
2. Description of the Related Art
Conventional forensic DNA specimens are commonly matched to alleged criminal suspects in modern law enforcement using human identification systems involving the amplification of highly polymorphic short tandem repeats (STRs) by polymerase chain reaction (PCR). PCR is a powerful tool which allows for replicating/amplifying trace amounts of DNA fragments into quantities that can be analyzed in a meaningful way. This technology has been adapted for DNA sequencing, DNA fingerprinting etc., and has the ability to detect specific DNA fragments in samples.
Thus, forensic DNA analysis is accomplished using the high power of discrimination and rapid analysis speed of STR markers in the human genome, and has now become the most popular method of choice in forensic DNA analysis.
By way of explanation, STRs are short stretches of DNA that arise when a pattern of two to six nucleotides are repeated and the repeated sequences are adjacent to each other. STR sequences vary in length of the repeat unit, number of repeats and in the rigor with which they conform to an incremental repeat pattern. STR markers are scattered throughout the genome and occur as frequently as one every 10,000 nucleotides. Based on 2001 International Human Genome Sequencing Consortium data, STRs or microsatellites account for approximately 3% of the total human genome. Currently 13 STRs are analyzed for human identification in forensic analysis, using well-known PCR methods.
Although STR analysis is commonly used, it suffers from several pitfalls, the most significant of which arises from contamination of the query DNA samples prior to STR analysis via PCR methods, and the time it takes to perform the PCR analysis.
For example, the DNA to be analyzed for STRs from sexual assault evidence should ideally come from the sperm of the assailant. However, the sperm sample is often commonly contaminated with (1) epithelial cells lining the vagina, and occasionally, with (2) epithelial cells from the mouth (buccal cells), and (3) cells from the skin, as well as cells in the urine sample. One might also expect to see erythrocytes, neutrophils, foam cells (non-descript epithelial cells), etc., in sexual assault crime scene samples as well.
Thus, it is clear that better and more accurate STR analysis will be achieved if the sperm cells could be separated from any or all of the contaminating cells before PCR is performed.
Commonly used methods like differential extraction cannot completely separate male (assailant) sperm and female (victim) epithelial cell DNA. For example, initial lysis using reductant free solution, lyses epithelial cells (the most common contaminant in a sexual assault forensic sample) and leaves sperm cells intact for effective separation of DNA fractions. However, differential lysis often causes immature sperm cell lysing. Therefore unwanted DNA often gets coamplified during PCR. This leads to mixed profile and incomplete STR analysis causing more than 50% of STR analysis based human identification to fail.
In addition, another limitation in solving forensic cases comes from the limited availability of cells for analysis. This may be due to limited evidence samples being present, degradation of the DNA and cell samples in general over time, and/or the presence of very few sperm cells in a sexual assault crime sample, to be able to solve the case based on standard PCR.
Thus, a method that would prevent or alleviate the above problems, and provide a fast, effective, and reliable method of sorting cells with single cell precision in a forensics sample, is desired.